Cellular proliferation and vascularization in ovine fetal ovaries: effects of undernutrition and selenium in maternal diet

Abstract

Sheep were fed a maintenance (M) diet with adequate (A) Se or high (H) Se concentration from 21 days before breeding to day 135 of pregnancy. From day 50 to day 135 of pregnancy (tissue collection day), a portion of the ewes from ASe and HSe groups were fed restricted (R; 60% of M) diet. Fetal ovarian sections were stained for: 1) the presence of proliferating cell nuclear antigen (a marker of proliferating cells) to determine the proportion of proliferating primordial follicles, or the labeling index (LI; percentage of proliferating cells) for primordial, primary, secondary and antral follicles, stromal tissues, and blood vessels; 2) factor VIII (a marker of endothelial cells) or 3) a presence of apoptotic cells/bodies. The number of proliferating primordial follicles and the LI of primordial follicles was decreased by R and/or HSe diets. The LI was similar for theca and granulosa cells, and for secondary or antral follicles, but was greater in secondary and antral than in primordial and primary follicles. R diet and/or Se affected the LI in all follicle types, in stromal tissues and blood vessels. A dense network of blood vessels was detected in the areas containing secondary to antral follicles, medulla, and hilus, but areas containing primordial follicles were poorly vascularized. The number of apoptotic cells was minimal. These results demonstrate that nutrient restriction and/or Se level in the maternal diet affected cellular proliferation in follicles, blood vessels, and stromal tissues in fetal ovaries. Thus, plane of nutrition and Se in the maternal diet may impact fetal ovarian development and function.

Placental development during early pregnancy in sheep: vascular growth and expression of angiogenic factors in maternal placenta

“…Image analysis was performed as previously described in detail (Borowicz et al 2007, Grazul-Bilska et al 2009). Images of randomly chosen areas of ICAR and CAR (5-10 per uterine section/sheep; 0.025 mm 2 per field) stained for PCNA or factor VIII were taken at 600!…”

Markers of ovarian antral follicular development in sheep: comparison of follicles destined to ovulate from the final or penultimate follicular wave of the estrous cycle

“…Detection of factor VIII, PCNA, and GJA1 was performed as described previously (Grazul-Bilska et al 1998, 2006, 2009, Pant et al 2005. Briefly, ovarian tissue sections were deparaffinized, rehydrated, and incubated with 3% H 2 O 2 in methanol to eliminate endogenous peroxidase activity.…”

“…The images were then used for quantitative image analysis using the Image-Pro Plus software (Media Cybernetics Inc., Silver Spring, MD, USA). Then, we determined the area that exhibited positive staining for factor VIII (occupied by endothelial cells) in the theca layer; or for GJA1 per tissue area for the granulosa and theca layers separately; and the LI (calculated as a percentage of proliferating cells out of the total cells per marked area of follicle) for the granulosa and theca cells separately, as described previously (Grazul-Bilska et al 2007, 2009). …”

First Transplantation of Isolated Murine Follicles in Alginate

“…Positive and negative cells present inside the alginate matrix were counted in order to calculate the proportion of proliferating cells. For follicular growth, follicles with at least one Ki67-positive GC were considered to be growing [14][15][16][17].…”